Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (8): 1739-1750.doi: 10.3864/j.issn.0578-1752.2021.08.013

• HORTICULTURE • Previous Articles     Next Articles

Genetic Diversity Evaluation of Castanea in China Based on Fluorescently Labeled SSR

NIE XingHua1(),ZHENG RuiJie2(),ZHAO YongLian3,CAO QingQin1,4,QIN Ling1,4,XING Yu1,4()   

  1. 1College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206
    2Liaoning Economic Forest Research Institute, Dalian 116000, Liaoning
    3Chestnut Technology Experiment and Extension Station of Huairou District, Beijing 102206
    4Advanced Innovation Center for Forest Molecular Design and Breeding, Beijing 102206
  • Received:2020-07-02 Accepted:2020-09-15 Online:2021-04-16 Published:2021-04-25
  • Contact: RuiJie ZHENG,Yu XING E-mail:niexinghuabua@163.com;zhengruijie2006@163.com;xingyubua@163.com

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Abstract:

【Objective】The characteristics of genetic diversity, relationships and population structure of Castanea plants in China were analyzed with SSR molecular markers, so as to provide a theoretical basis for variety improvement, germplasm innovation and utilization of Castanea plants. 【Method】Firstly, 330 SSR molecular markers were screened among 12 chestnut cultivars from different production areas, and 12 pairs of high-quality SSR primers were obtained from them. Secondly, 96 accessions of Castanea from 4 species were detected by high resolution capillary electrophoresis. Finally, Power Marker 3.25, GenAlEx 6.51, FigTree v1.4.3 and Structure 2.3.3 were used to analyze the population genetic diversity of all accessions. 【Result】A total of 129 alleles were acquired from 96 accessions, and the average variation of each marker was 10.750. The gene diversity (GD) ranged from 0.656 (CmSI0396) to 0.877 (CmSI0930) with an average of 0.800; the observed heterozygosity (Ho) ranged from 0.329 (CmSI0742) to 0.769 (CmSI0702) with an average of 0.615; the expected of heterozygosity (He) ranged from 0.489 (CmSI0742) to 0.789 (CmSI0922), with an average of 0.672; the polymorphic information content (PIC) ranged from 0.586 (CmSI0396) to 0.868 (CmSI0930), with an average of 0.774. Among different Castanea species, in terms of number of alleles (Na), number of effective alleles (Ne) and Shannon diversity index, Castanea seguinii had the highest genetic diversity, followed by Castanea mollissima, and Castanea crenata was the lowest. According to pairwise population Fst values, the genetic differentiation value of Castanea species was 0.077-0.180, showing moderate to high differentiation among the species. Compared with Castanea crenata, both Castanea mollissima and Castanea henryi exhibit relatively high genetic differentiation with Fst values of 0.165 and 0.180, respectively. At the same time, the gene flow (Nm) of the Castanea plants was 1.580>1, suggesting frequent gene exchange among Castanea plants, and which therefore reduced the degree of genetic differentiation among the species caused by genetic drift. The results of analysis of molecular variance (AMOVA) showed that the variation mainly occurred within populations, accounting for 73% of the total variation, and the variation among populations made up 27%. The results of UPGMA cluster analysis, principal coordinate analysis and population genetic structure were consistent, and the genetic background of each accessions had obvious inter-species boundaries. Some accessions inherited the genetic information of different ancestor species in generational inheritance. For example, accession 65, 71 and 82 are mixed types, which contain the genetic background of Castanea seguinii and Castanea crenata, but there is geographic isolation between the two species. Castanea plants in the same ecological region existed extensive gene exchange, and there were no complete reproductive isolation. Accession 48 (‘Guangdongaisheng’) had the genetic background of both Castanea mollissima and Castanea seguinii. In terms of geographical distribution, the native place of this accession was in the overlapping ecological area of Castanea mollissima and Castanea seguinii. 【Conclusion】The 12 pairs of SSR primers screened could accurately assess the genetic diversity of Castanea plants in China. Comprehensive cluster analysis could confirm that the classification of Castanea plants was highly consistent with the inter-species information and there was a certain amount of gene exchange between species.

Key words: SSR, Castanea, cluster analysis, population genetic structure, population genetic differentiation

Table 1

Geographical distribution of the selected materials"

资源分类Germplasm type 总数量Accession amount 来源Origin 数量Number
板栗种群POP 1 50 中国陕西 Shaanxi, China 6
中国北京 Beijing, China 11
中国河北 Hebei, China 3
中国山东 Shandong, China 10
中国湖北 Hubei, China 10
中国云南 Yunnan, China 3
中国浙江 Zhejiang, China 4
中国江苏 Jiangsu, China 2
中国广东Guangdong, China 1
锥栗种群POP 2 13 中国福建 Fujian, China 13
茅栗种群POP 3 19 中国湖南Hunan, China 7
中国江西 Jiangxi, China 1
中国湖北 Hubei, China 2
中国安徽 Anhui, China 9
日本栗种群POP 4 14 中国辽宁/日本 Liaoning, China/Japan 14
合计 Total 96

Table 2

The information of 12 SSR markers"

序号
Number		 标记名
Marker ID		 DNA序列号
GenBank ID		 上游引物
Forward primer (5′-3′)		 下游引物
Reverse primer (3′-5′)		 观察基序
Repeat motif
1 CmSI0396 290474606 AACTCCCACCACTCACATCC TTTCGGACCATCCAGAACTC CACACC
2 CmSI0561 290474702 CGTATAGGGTGGAAACGGAA GGACAAGCAAATCACGGAAT TCG
3 CmSI0614 290476556 TTGTGGTGAAGCTGACATCG GGGTACTACCACAACATGCAG GTT
4 CmSI0658 290474781 AAAACGGTTTGTGGTGAAGC GCCAACCAGTCAAGGGTACT GTT
5 CmSI0702 290474818 GAAACACACCAGAGAGATGCAG TTTTATACAGAGACATACTATCCTACACAG TC
6 CmSI0742 290474850 GACGCTCCTCAGCTTTTGAC TGCCGGTCAATTCTTCTTCT AG
7 CmSI0800 290474899 TTATGGCAACCCTCCTGTTT CTGAAATGATCGATGCTGCT TC
8 CmSI0853 290474950 GGAGGAGGAGGAGCTCATTG CCTTGGAGAGCTGCCAGTAG TCT
9 CmSI0871 290474967 AGGGGGTGGAAGAACCTATG AGATTGCAAGTGGGGAATTG TCT
10 CmSI0883 290476060 CAGCATCAGCACTCGTTCA GGGATTGAGAGGATGAAGCA AGC
11 CmSI0922 290475011 AATCTGAACCCCTCCGATCT ACCAACAACATGTGCCAAAA TTG
12 CmSI0930 290475019 CCATTTAGCATGCATAGTCATACC GCAAGGATGTAGGTCGAATCA ATAC

Fig. 1

Alleles of high polymorphism primers at CmSI0561 and CmSI0614 in 12 Chinese chestnut cultivars"

Table 3

The key genetic statistics of 12 SSR markers"

标记名
Marker		 主要位点频率
MAF		 位点数
Na		 位点多样性
GD		 观察杂合度
Ho		 期望杂合度
He		 多态信息含量
PIC		 遗传分化指数
Fst		 基因流
Nm
CmSI0396 0.390 4 0.656 0.552 0.544 0.586 0.190 1.063
CmSI0561 0.427 9 0.739 0.533 0.527 0.708 0.257 0.724
CmSI0614 0.183 14 0.839 0.563 0.740 0.818 0.130 1.667
CmSI0658 0.224 8 0.829 0.721 0.722 0.805 0.103 2.176
CmSI0702 0.302 11 0.806 0.769 0.772 0.782 0.096 2.351
CmSI0742 0.422 7 0.717 0.329 0.489 0.673 0.304 0.571
CmSI0800 0.391 12 0.791 0.591 0.706 0.771 0.146 1.457
CmSI0853 0.287 11 0.834 0.696 0.764 0.814 0.066 3.534
CmSI0871 0.339 12 0.788 0.714 0.636 0.760 0.255 0.730
CmSI0883 0.240 13 0.867 0.652 0.682 0.853 0.221 0.880
CmSI0922 0.237 11 0.860 0.701 0.798 0.845 0.079 2.920
CmSI0930 0.260 17 0.877 0.561 0.686 0.868 0.220 0.885
平均值 Mean 0.308 10.750 0.800 0.615 0.672 0.774 0.172 1.580

Fig. 2

Comparison of silver staining results for polyacrylamide gel electrophoresis and detection results for capillary electrophoresis"

Table 4

The key genetic statistics of genetic diversity of different Castanea plant populations"

群体
Pop		 样品数
Sample		 变异位点数
Na		 有效等位变异
Ne		 Shannon多样性指数
I		 观察杂合度
Ho		 期望杂合度
He		 无偏预期杂合度
UHe
板栗种群 Pop1 50 6.667±0.732 3.708±0.317 1.453±0.086 0.648±0.024 0.707±0.026 0.714±0.026
锥栗种群 Pop2 13 5.417±0.288 3.141±0.213 1.343±0.056 0.633±0.065 0.665±0.022 0.693±0.023
茅栗种群 Pop3 19 7.083±0.883 4.748±0.712 1.587±0.158 0.623±0.047 0.719±0.047 0.739±0.048
日本栗种群 Pop4 14 5.333±0.620 3.080±0.416 1.219±0.145 0.556±0.072 0.598±0.058 0.621±0.061

Table 5

Pairwise population Fst values"

板栗种群
Pop1		 锥栗种群
Pop2		 茅栗种群
Pop3		 日本栗种群
Pop4
0.000 板栗种群 Pop1
0.077 0.000 锥栗种群 Pop2
0.089 0.115 0.000 茅栗种群 Pop3
0.165 0.180 0.108 0.000 日本栗种群 Pop4

Fig. 3

Distribution of Delta K values for structure analysis in the tested Castanea plants"

Fig. 4

Population structure diagram of the tested Castanea plants"

Table 6

AMOVA analysis of different Castanea populations"

变异来源
Source of variance		 自由度
df		 方差总和
SS		 平均方差
MS		 变异组分
Variance component		 变异百分率
Variation (%)		 P*
种群间 Among Pops 3 252.554 84.185 3.589 27% <0.01
种群内 Within Pops 92 878.457 9.548 9.548 73% <0.01
总量 Total 95 1131.010 13.137 100%

Fig. 5

Principal coordinate analysis (PCoA) of the tested Castanea plants"

Fig. 6

UPGMA dendrogram of the tested Castanea plants"

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